How to do anti-cancer activity of synthesized compounds on cancer cell lines.
Or How to screen the samples for anti-cancer activity.
I recently learned and performed the anti-cancer activity (or cell proliferation assay or anti-cell proliferation assay) of synthesized molecules on different cancer cells. I am sharing the procedure we followed and is mentioned here.
Material Required:
1. Cancerous cells 2. Culture media 3. Dye
Instruments
Microscope, Cnetrifuge, Cell counter, plate reader (absorbance spectrometer), CO2 incubator, cell-culture hood.
Apparatus
Pipettes, tips, petridishes, 96 well plates.
Procedure:
A. Cell culture (Growing the cells):
The first step is to grow the cell in culture media. You can purchase cancerous cells from local cell repository /or supplier. Cells are generally supplied/stored (Dormant cell) in liquid nitrogen (in small ampules). You will have to select the cell type for which cancer type you want to perform the activity study (Note1).
1. Take out the Dormant cells (ampule) from storage (liquid nitrogen) and keep in hood (sterile/cell culture).
Perform these steps inside the sterile/cell culture hood.
2. Prepare the cell culture media (Note2). (If already prepared, then keep at 37oC water bath for 10-15 minute before use).
3. Add culture media (10-12 ml) to petridish (apparatus etc. may be rinsed with 70% ethanol solution before taking to hood).
4. Dormant cell (stored in liquid nitrogen/taken out in step1) are incubated at 37oC (water bath) for one minute and transferred/added cells to above petridish (having culture media).
In CO2 incubator
5. Petridish is kept in CO2 incubator at 37oC for cell growth.
inside hood
6. The culture medium of the growing cells can be changed after 72 hours or so with the fresh media (Note3).
7. Observe the cells on transmission microscope for the uniformity, dead cells (floating ones), any contamination.
B. Cell proliferation assay:
7. When sufficient cells are there, remove the media from plate. Add trypsin solution (Note 4) (~ 5ml), incubate at 37oC for 5 min, observe under microscope to see nearly all the cells are floating, add media to quench trypsin.
8. Transfer to centrifuge tube and centrifuge at ~ 1000 rpm (Note 5).
9. Decant the supernatant, add media (10 ml) and vertex to disperse the cells.
10. Cell Counting: take 1.8 ul of cell solution, add .2 ul Trypan dye and transfer on cell counter plate and do the recording on cell counter (with dilution factor of 1.1). This gives the number of cells present per 1ml of cell solution.
11. Dilute the solution with media in such way that 100ul of solution has nearly ~5000 cells (Note6).
12. Now take the 96 well plate and add 100 ul of cell solution to each well. The no. of well to be used depend upon the number of samples/compounds to be screened (Note7).
13. Incubate the plate for 24h (give sufficient time to cell to adhere to walls and grow) and then 20 ul of 300uM solution of each compounds (in 3 wells for each samples for triplicate study). [in case of leukemia/blood cells, the samples are added after 1 hr of plating .. as that need not be grown for 24 hrs and are already taken in sufficient amount). Make the 3-4 plates for reading at different time interval (generally 24, 72 and 96 h).
14. Now keep the plates in incubator (37oC, 5% CO2).
15. After 24h (or 72h or 96h), take the plate, add formazone forming dye (20ul of solution) to the well to be read. Incubate for 1h (2-3 h for leukemia cells).
16. Now record the reading (absorbance at 490nm) at 96 plate reader.
C. Calculations
1. Take the avarage of set of 3 reading for each samples (and measure standard deviation). Keeping the negative control reading as 100%, calculate the percentage for the compounds (with standard deviation).
This gives you cell proliferation in respect of control. If in case of of compound A, the cell proliferation was 45%, then it means cell proliferation was inhibited by 55%.
This protocol is used for preliminary screening of samples/compounds at 50 uM. If samples are found sufficient active, then further IC50 is calculated by doing the similar study at different concentrations.
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Note1. We used Ovarian cancer cells (SK-OV-03), Colon Cancer cells (HT-29), Breast Cancer cells (MDA-MB-468) and leukemia cells (CCRF-CEM). For one study, our molecules (designed and synthesized) were supposed to be Src kinase inhibitors and this enzyme is over expressed in first 3 cell lines (Ovarian, colon and breast), so were selected these for the study. For another study, we used all cell lines.
Note2. Composition of culture media: It varies from cell to cell type.
Note3. The cells generally adhere to walls of petridish, so just tilt the plate, remove used media with pipette and add fresh media (10-12 ml). The Blood cells (like leukemia cells (CCRF)) do not adhere to surface, so their media can be changed by centrifuging, removing upper part (media) and replace with new media.
Note4. Trypsin solution is used to remove the cell adherance from wall. Plate is incubated for approx. 5-15 minute at 37 oC (check under microscope so that nearly all the cells are floating). Longer time incubation should be avoided to prevent cell degradation.
Note5. ~1000 rpm is optimum (otherwise generally it is done at 2500 to 3000 rpm) to avoid the centrifugation of dead cells. If see more dead cell, then redissolve the pallete in media and centrifuge again.
Note6. This is not fix. you can take any number of cell with a general count used ~3000 - ~5000. In case of leukemia cells, it should be ~25,000 to ~50,000.
Note7. Like if you want to screen 5 compounds, then add one for negative control (having no additive/compound) and one for positive control (if you are using any standard drug for reference). For triplicate study of each sample, multiply with 3.
Note8. Make the solution of compounds (DMSO+water) in such a way that 20 ul of solution has 50 uM concentration. DMSO amount should not be more that 5% in solution (1-2% in media).
If you have any suggestion or correction, then let me know, I will update this note accordingly.
